Wang, Peng et al. (2011) International Worm Meeting "Caenorhabditis elegans O-GlcNAc cycling mutants alter the proteotoxicity of models of human neurodegenerative disorders."

Forsythe, Michele, Wang, Peng, Lazarus, Brooke, Hanover, John, Love, Dona, Krause, Michael

[International Worm Meeting, 2011]

O-linked N-acetylglucosamine (O-GlcNAc) addition is an important post-translational modification that occurs on hundreds of proteins, including nuclear pore proteins, transcription factors, proteasome components and neuronal proteins. O-GlcNAc can be added onto and removed from serine or threonine residue by two evolutionally conserved enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. O-GlcNAcylation is abundant in the brain and it has been linked to human neurodegenerative disease. We have exploited viable null alleles of the enzymes of O-GlcNAc cycling to examine the role of O-GlcNAcylation in well-characterized C. elegans models of neurodegenerative proteotoxicity. O-GlcNAc cycling dramatically modulated the severity of the proteotoxic phenotype in transgenic models of tauopathy, b-amyloid peptide and polyglutamine expansion. Intriguingly, loss-of-function of OGT alleviated, while loss of OGA enhanced these proteotoxicity phenotype. Consistent with these observations, the O-GlcNAc cycling mutants exhibit altered stress responses and changes in the protein degradation machinery. These findings suggest that modulators of O-GlcNAc cycling may prove useful for anti-neurodegenerative disease therapies.

Wamucho, A. et al. (2017) International Worm Meeting "Investigating multigenerational effects of silver nanoparticles in Caenorhabditis elegans: A Toxicogenomic Approach."

Wamucho, A., Tsyusko, O., Unrine, J.

[International Worm Meeting, 2017]

Studies in the nematode Caenorhabditis elegans have shown that exposure to as-synthesized and transformed (sulfidized) silver nanoparticles (sAg-NPs) can cause various toxic effects, but genomic effects that can be induced by prolonged exposure over multiple generations have not been investigated. A previous multigenerational study of continuous exposure of C. elegans to AgNO3, Ag-NPs and sAg-NPs showed that increased sensitivity, in terms of reproductive toxicity from Ag, occurred as early as the second generation for AgNO3 and Ag-NPs, but not sAg-NPs. This suggested that Ag-NPs may cause mutations or epi-mutations. In this study, we used wild type N2 C. elegans as a model organism to determine if mutations and/or epi-mutations contribute to the observed multigenerational effects. Exposure was carried out using sub-lethal concentrations (EC30 for reproduction) of AgNO3, Ag-NPs and sAg-NPs in simulated soil pore water for 10 generations with the parent generation (F0) unexposed for all groups. Four replicates were used per treatment, from which DNA was extracted from an entire brood of a single worm for whole genome DNA sequencing by NextSeq. Analysis of sequencing data revealed no significant differences in the total number of mutations (SNPs, deletions or insertions) from the controls. This suggests that induced germline mutations may not be responsible for the increased sensitivity observed in multigenerational exposures. We hypothesized that epigenetic mechanisms are involved. Increased DNA methylation (6-methyladenine), an epigenetic marker, which was recently discovered in C. elegans, has been shown to negatively impact reproduction over multiple generations. We are currently investigating DNA methylation as a potential mechanism by which reproductive toxicity can be passed on to subsequent generations after exposure to Ag-NPs. Preliminary experiments using slot blots show that there is a potential increase in DNA methylation after 2 generations of exposure to pristine Ag-NPs. Interestingly, there seems to be a decrease in DNA methylation for AgNO3 exposed worms. In addition to confirming these results and testing sAg-NPs, we will also examine if the higher levels of DNA methylation persist in subsequent generations after cessation of exposure.

Wamucho, A. et al. (2015) International Worm Meeting "Investigating multigenarationaleffects of silver nanoparticles on reproduction and genomic instability in C. elegans."

Wamucho, A., Tsyusko, O., Unrine, J.

[International Worm Meeting, 2015]

The increasing use of silver nanoparticles (AgNP) in consumer products greatly increases the potential for the environmental release of these particles. Studies have shown that exposure of the Caenorhabditis elegans to NP has adverse effects. Decreased reproduction, alteration in gene expression, and increased mortality has been observed in different studies involving the exposure of C. elegans to AgNPs . Some of these effects have been shown to be multigenerational. The short generation time for C. elegans makes it an ideal model for studying multigenerational effects of silver NP. In this study, C. elegans were exposed to AgNP in synthetic soil pore water continuously over 10 generations at an environmentally relevant concentration. Effects on reproduction were measured by counting the offspring of individual nematodes. Individual variability at five microsatellite loci was also assessed using PCRs and DNA fragment analysis. AgNP release silver ions into exposure medium which accounts for some toxicity. Biotransformation such as sulfidation of AgNP occurs in the environment. AgNO3 and sulfidized AgNP were used as controls. No effects on reproduction were observed over 10 generations. There were also no observed differences for the microsatellite loci that were tested. This was probably due to the low concentration that was used to mimic environmental concentrations of NP and/or selection of the loci used for this experiment. A preliminary experiment involving the continuous exposure for over five generations has been carried out using EC30 (1.3mg/L) for reproduction. This is a much higher concentration compared to the concentration used in the previous experiment (0.3mg/L). Unlike the previous experiment, both control and exposed generations originated from the same lineage. DNA extractions were carried out from an entire brood of a single nematode. Whole genome sequencing was carried out using MiSeq. Sequencing data obtained from the F0 and F5 generations for control and exposed nematodes were aligned to a reference genome. A conservative approach was used for mutations' detection using SnpEff program. Mutations involved missense, deletion and insertions. The mutations were observed in coding, regulatory 5' and 3' untranslated regions, intergenic, introns, and microsatellite regions. Mutations within coding regions as well as immediately upstream and downstream of coding regions will be examined. Some of these mutations may have consequences in the function of proteins as well as regulation of gene expression. A full scale experiment, involving all of the treatments discussed for the first experiment will be carried out to further examine multigenerational effects at the genomic level.

Lee, J. et al. (2009) International Worm Meeting "Proteomic analysis of O-GlcNAcylation during dauer formation in C. elegans."

Paik, Y., Kim, K., Lee, J.

[International Worm Meeting, 2009]

Modification of proteins at serine or threonine residues with N acetylglucosamine (O-GlcNAcylation) plays a regulatory role in many model organisms. Here, we investigated the mechanism by which O-GlcNAcylation regulates entry into the stress-induced dormant state dauer in Caenorhabditis elegans. We confirmed that ogt-1 (O-GlcNAc transferase) mutants exhibited a dauer-defective phenotype whereas oga-1 (O-GlcNAcase, catalyzes O-GlcNAc removal) mutants exhibited a dauer-prone phenotype when treated with daumone. Consistent with these findings, treatment with low levels of daumone and the O-GlcNAcase inhibitor PUGNAc enhanced the frequency of dauer entry. Treatment of daf-2 with PUGNAc increased the frequency of dauer entry, providing additional evidence that O-GlcNAcylation promotes dauer formation. Alterations to the proteome as a result of induction of O-GlcNAcylation were analyzed by two-dimensional electrophoresis (2DE) using lysates of N2 and oga-1 worms. Seven differentially expressed protein spots were further analyzed using LC-MS/MS. The identities of these proteins suggest that O-GlcNAcylation influences stress resistance, protein folding, and mitochondrial function. To identify potential target proteins that are O-GlcNAcylated during dauer formation, we specifically labeled O-GlcNAc with fluorescent dye using O-GlcNAc labeling system containing TAMRA fluorescence dye targeting O-GlcNAcylated proteins. The fluorescently labeled samples were resolved by 2DE and 20 protein candidates were selected. Our data suggest that O-GlcNAcylation may regulate a shift in some proteins including cytoskeletal and protein turnover during dauer formation. Thus, these data may be valuable in identifying the mechanism by which high levels of O-GlcNAcylation enhance dauer formation. (This study was supported by a grant from the Korea Health 21 R&D project, Ministry of Health and Welfare of Republic of Korea [A030003 to YKP].).

Mahaffey, M. et al. (2017) International Worm Meeting "O-GlcNAc cycling and mitochondrial function."

Mahaffey, M., Austin, B., Berninsone, P., Sacoman, J.

[International Worm Meeting, 2017]

O-linked- beta -N-acetylglucosamine (O-GlcNAc) modified proteins are critical in myriad cellular processes and functions. Abnormal O-GlcNAcylation is associated with a spectrum of diseases, including Alzheimer's disease, cancer, cardiovascular disease, and diabetes. Mitochondrial protein O-GlcNAcylation is emerging as a key regulator of cellular energetic metabolism, redox signaling and cell survival pathways, but the mechanisms involved are largely unknown. O-GlcNAc transferase (OGT) is the enzyme responsible for the addition of O-GlcNAc to target proteins while O-GlcNAcase (OGA) catalyzes the removal of the modification from target proteins. OGT and OGA are encoded by single genes in C. elegans (ogt-1 and oga-1, respectively). OGT and OGA modulate lifespan, stress susceptibility and oxidative stress resistance. We hypothesize that O-GlcNAc cycling mediated by OGT and OGA plays a role in regulating mitochondrial metabolism and morphology. To that end, we measured oxygen consumption rate (OCR), a proxy for mitochondrial oxidative phosphorylation function, in N2, ogt-1 and oga-1 null mutants. OCR was measured using the Seahorse Bioscience XFe 24 Extracellular Flux Analyzer and normalized by number of worms for each individual well. Our results suggest that altered O-GlcNAc cycling reduces oxygen consumption by negatively impacting mitochondrial oxidative metabolism. In addition, O-GlcNAc cycling mutants have altered sensitivity to rotenone, an inhibitor of NADH ubiquitone reductase (Complex I). These results suggest that modulation of mitochondrial function by O-GlcNAc cycling may underlie some of the phenotypes of O-GlcNAc cycling mutants.

Scott Everet Baird et al. (2004) East Coast Worm Meeting "Association of Oscheius species with millipedes in the Wright State University Woods."

Scott Everet Baird, Christine M Spice

[East Coast Worm Meeting, 2004]

Rhabditid nematodes commonly form phoretic or necromenic associations with other soil invertebrates. These associations typically are as dauers although although associations of other larval stages also have been reported. Two hermaphroditic species of Oscheius , O. myriophila and an isolate tenatively identified as O. necromena , have been reported as necromenic associates of millipedes. Both of these species have been found in association with the millipede Pseudopolydesmus serratus in the Biology Preserve on the campus of Wright State University. A third as yet unnamed hermaphroditic species of Oscheius was found in association with a second millipede, Oxidus gracilis . O. myriophila , O. necromena , and O. sp. were found at the same collection site, i.e. they were sympatric. O. myriophila never was found in association with Ox. gracilis despite this millipede being its type host. When first observed, all collected nematodes were L4s, consistant with a previous report of the association of O. myriophila L4s with millipedes. Relationships among these species were investigated through sequence comparisons of 18S rDNA. O. myriophila , O. necromena , and O. sp. clustered together as a monophyletic clade within the insectivora -group of Oscheius . Thus, association with millipedes and their hermaphroditic mode of reproduction may be ancestral characters of this group of species.

Rahman, Mohammad M. et al. (2009) International Worm Meeting "Elevated O-GlcNAc modification can extend Caenorhabditis elegans lifespan."

Rahman, Mohammad M., Wells, Lance, Karim, Enas, Stuchlik, Olga, Kipreos, Edward

[International Worm Meeting, 2009]

O-linked beta-N-acetylglucosamine (O-GlcNAc) modification is an abundant nucleo-cytoplasmic post-translational glycosylation of proteins associated with age-related diseases like Alzheimer''s, Parkinson''s, and type II diabetes. However a link between O-GlcNAc modification of proteins and organismal aging has not been demonstrated. This work uses the nematode C. elegans to establish a link between nutrient availability, O-GlcNAc cycling, and longevity. We found that O-GlcNAc modification of protein(s) is critical for normal lifespan in adult animals, while unchecked O-GlcNAc modification of protein(s) increases adult lifespan in C. elegans. We demonstrate that the adult lifespan extension arising from an elevated level of O-GlcNAc modification is dependent on the DAF-16/FoxO transcription factor. DAF-16 is a key factor in insulin-like signal transduction, which regulates reproductive development, lifespan, and dauer formation in response to nutrient availability. Our current data indicates that O-GlcNAc cycling influences a subset of insulin-like signaling-mediated functions that regulate adult lifespan, without affecting other downstream aspects of the insulin-like signaling pathway.

Taub, D.G. et al. (2019) International Worm Meeting "O-GlcNAc signaling enhances neuronal regeneration through modulation of cellular metabolism."

Awal, M.R., Taub, D.G., Gabel, C.V.

[International Worm Meeting, 2019]

The lack of neuronal regeneration after damage to the central nervous system remains a critical deficiency in modern medicine. While regrowth of a severed axon is inherently energetically demanding, the mechanisms of metabolic regulation in a damaged neuron remain largely unknown. This represents a critical gap in our understanding of neuronal repair that is fundamental to therapeutic strategies. We have recently discovered that alterations in O-GlcNAc signaling can shift cellular metabolism to dramatically potentiate the regenerative capacity of a damaged neuron in vivo. O-linked ?- N-acetylglucosamine (O-GlcNAc) is a post-translational modification of serines/threonines that functions as a sensor of cellular nutrients. Performing in vivo laser axotomies in C.elegans, we find that neuronal regeneration is substantially increased by disruptions of either the O-GlcNAc Transferase or the O-GlcNAcase that decrease and increase O-GlcNAc levels, respectively. A lack of O-GlcNAc acts through the AKT-1 branch in the insulin-signaling pathway to utilize glycolysis. In contrast, increased O-GlcNAc levels activate an opposing branch of the insulin-signaling pathway whereby SGK-1 modulates the FOXO transcription factor DAF-16 to influence mitochondrial function. Exploiting the effects of O-GlcNAc signaling on metabolism and regeneration, we are determining the metabolic response within a damaged neuron, how cellular metabolism acts as limiting factor in neuronal regeneration and how it might be altered for neuro-therapeutic benefits.

Carta LR et al. (1995) International C. elegans Meeting "NEW HERMAPHRODITIC SPECIES OF OSCHEIUS (ANDRASSY, 1976) (RHABDITIDAE, NEMATODA)"

Thomas WK, Carta LR, Sternberg PW

[International C. elegans Meeting, 1995]

Morphological, life cycle and molecular characteristics are used to describe seven hermaphroditic species of Oscheius (Andrassy, 1976) including O. brevesophaga, n. sp. O. mesoesophaga n. sp., O. fijiensis n. sp., O. spiculunca n. sp. O. longirecta n. sp., O. subvulva n. sp., and O. longimascula n. sp. These were compared with other living isolates of the group provided through David Fitch. Starvation induced males were mated among the isolates to define distinct biological species. These species were sequenced over a 310 nucleotide bp region of the nuclear 28S rRNA. Vulval anatomy was observed in the early L4 stage. Life cycle was determined from egg to egg at 20 C. to within half a day. The only non-fertile hybrids produced were those of O. mesoesophaga hermaphrodites mated with O. tipulae males. With just 1 base pair difference between each sp. in the series, O. brevesophaga, mesoesophaga and tipulae are best distinguished by esophageal length. By contrast, three interbreeding isolate groups of O. brevesophaga are defined by 1 or 2 bp changes. Thus, at least 2 bp changes may be necessary to distinguish a biological species in this group, while no change may be sufficient (e.g. as in Rhabditis anomala and a new hermaphroditic sibling species, Rhabditis heranamaloides). This can help define species where males are nearly impossible to find or mate. Vulval anatomy revealed that 2o lineages generated 4 cells and 3o generated 4 cells (4:4) in the three non- Dolichura groups, and 4:2 cells in three of the four species of the Dolichura group examined. (Caenorhabditis has a 7:2 pattern.) A significant number of O. subvulva individuals showed an interesting asymmetry with 4 cells resulting from the 3o P4.p lineage, while the 3o P8.p lineage gave only two cells. Four major groups are defined by larger base pair difference gaps between than within groups. These correspond to somewhat distinct groups morphologically and developmentally as follows: Tipulae group: Spicules <30 micron with tips not visibly swollen, Life cycle < 5 days at 20 C, 2o:3o = 4:4. (O. brevesophaga, mesoesophaga, tipulae) Insectivora group: Spicule tips hooked or acute, Spicule > 30 micron, Bursa usually pseudopeloderan, >= 4 lateral lines, life cycle < 5 days at 20 C, 2o3o = 4:4. (O. longirecta, spiculunca) Fijiensis group: Spicule tips swollen, life cycle < 5 days at 20o C, Medial vulva, 2o:3o = 4:4. (O. fijiensis) Dolichura group: Spicules with swollen tips, Life cycle > 5 days at 20o C, Vulva usually > 50%, 2o:3o usually = 4:2. (O. dolichura, dolichuroides, subvulva, longimascula)

Eustice, Moriah et al. (2015) International Worm Meeting "O-GlcNAc cycling plays a crucial role in germline plasticity and acts in coordination with fatty acid beta-oxidation to regulate ARD entry in C. elegans."

Hanover, John, Eustice, Moriah

[International Worm Meeting, 2015]

Adult reproductive diapause (ARD), or the oogenic germline starvation response, is triggered when C. elegans are starved at the mid-L4 stage of development. ARD is characterized by extended lifespan and reproductive potential as well as germline plasticity, wherein the germline shrinks back to approximately 35 germ cell nuclei per gonad arm during starvation but regenerates following refeeding. Previous studies found that the fatty acid beta-oxidation protein NHR-49 plays a key role in the establishment of ARD. However, questions remain about the way in which changes in metabolism are sensed and trigger the plasticity observed with adult diapause.To address these questions, we aimed to examine the complex relationships between ARD and metabolism, specifically focusing on the modification O-N-acetyl-glucosamine (O-GlcNAc). O-GlcNAc is a dynamic monosaccharide post-translational modification that integrates signals from several metabolic pathways, including glucose and fat metabolism. Key enzymes in the cycling of O-GlcNAc are O-GlcNAc transferase (OGT), which is responsible for the addition of O-GlcNAc, and O-GlcNAcase (OGA), which removes the modification. Due to the fact that the substrate of OGT, UDP-GlcNAc, is produced as the final step of the hexosamine biosynthetic pathway, the regulation of O-GlcNAc cycling is uniquely situated to link metabolic signaling with growth, development, and longevity. Using mutants of OGT and OGA we discovered that O-GlcNAc cycling plays a prominent role in both ARD entry and exit. Furhter, we found that the role of O-GlcNAc in the establishment of ARD is genetically linked to NHR-49 function, such that impairment of O-GlcNAc cycling suppresses the NHR-49 entry defect.These findings suggest that perturbation of nutrient-sensitive O-GlcNAc is crucial to the developmental dynamics observed in ARD and acts in concert with NHR-49 to regulate entry into reproductive diapause. Understanding ARD dynamics is important as it provides a unique model in which to explore the relationship between nutrient status and the regulation of reproduction and aging. .